Polynucleotides and their use for detecting resistance to streptogramin A or to streptogramin B and related compounds

ABSTRACT

The present invention pertains to polynucleotides derived from staphylococcal genes encoding resistance to streptogramin A or to streptogramin B and chemically related compounds. This invention also relates to the use of the polynucleotides as oligonucleotide primers or probes for detecting Staphylococcal strains that are resistant to streptogramin A or to streptogramin B and related compounds in a biological sample. In another embodiment, the present invention is directed to the full length coding sequences of the staphylococcal genes encoding for resistance to streptogramin A or to streptogramin B from  Staphylococcus  and to the polypeptides expressed by these full length coding sequences. Further, this invention relates to the use of the expressed polypeptides to produce specific monoclonal or polyclonal antibodies that serve as detection means in order to characterize any staphylococcal strain carrying genes encoding resistance to streptogramin A or to streptogramin B.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application hereby claims the benefit under 35 U.S.C. §119(e) ofU.S. provisional application Ser. No. 60/050,380, filed Jun. 20, 1997.The entire disclosure of this application is relied upon andincorporated by reference herein.

BACKGROUND OF THE INVENTION

The present invention pertains to polynucleotides derived fromstaphylococcal genes encoding resistance to streptogramin A or tostreptogramin B and chemically related compounds. This invention alsorelates to the use of the polynucleotides as oligonucleotide primers orprobes for detecting Staphylococcal strains that are resistant tostreptogramin A or to streptogramin B and related compounds in abiological sample.

In another embodiment, the present invention is directed to the fulllength coding sequences of the staphylococcal genes encoding forresistance to streptogramin A or to streptogramin B from Staphylococcusand to the polypeptides expressed by these full length coding sequences.

Further, this invention relates to the use of the expressed polypeptidesto produce specific monoclonal or polyclonal antibodies that serve asdetection means in order to characterized any staphylococcal straincarrying genes encoding resistance to streptogramin A or tostreptogramin B.

The present invention is also directed to diagnostic methods fordetecting specific strains of Staphylococcus expected to be contained ina biological sample. The diagnostic methods use the oligonucleotideprobes and primers as well as the antibodies of the invention.

Streptogramins and related compounds (antibiotics) produced bystreptomycetes can be classified as A and B compounds according to theirbasic primary structures (Cocito, 1979). Compounds of the A group,including streptogramin A (SgA), pristinamycin IIA (PIIA), virginiamycinM, mikamycin A, or synergistin A, are polyunsaturated cyclicmacrolactones. Compounds of the B group, including streptogramin B(SgB), pristinamycin B (PIB), virginiamycin S, mikamycin B, andsynergistin B, are cyclic peptidic macrolactones (Cocito, 1979).Compounds of both groups, A and B, bind different targets in thepeptidyltransferase domain of the 50S ribosomal subunit and inhibitprotein elongation at different steps (Aumercier et al., 1992; DiGiambattista et al., 1989).

A decrease in the dissociation constant of PIB is observed in thepresence of PIIA because this latter antibiotic provokes aconformational modification of the bacterial ribosome at the bindingsites of these molecules. Thus, A and B compounds, which arebacteriostatic when used separately, act synergistically where combinedand become bactericidal, mainly against Gram-positive bacteria.

Natural mixtures such as pristinamycin (Pt), synergistin, virginiamycinand mikamycin, are used orally and topically. A semi-syntheticinjectable streptogramin, RP59500, consisting of a mixture ofderivatives of A and B compounds (Dalfopristin and Quinupristin,respectively) is currently undergoing in vivo experimental and clinicaltrials (J. Antimicrob. Agents Chemother. 30 (Suppl. A), entire volume,1992; Entenza et al., 1995; Fantin et al., 1995; Griswold et al., 1996;Torralba et al., 1995). Staphylococcal resistance to synergisticmixtures of A and B compounds (Pt MIC ≧2 μg/ml) is always associatedwith resistance to A compounds (PIIA MIC ≧8 μg/ml), but not necessarilywith resistance to B compounds (Allignet et al., 1996).

To date, four genes encoding resistance to A compounds have beenisolated from staphylococcal and enterococcal plasmids. The genes vat(Allignet et al., 1993), vatB (Allignet and El Solh, 1995), and satA(Rende-Fournier et al., 1993) encode related acetyltransferases(50.4-58.3% amino acids), which inactivate streptogramin A and similarcompounds. The staphylococcal gene vga (Allignet et al., 1992) encodesan ATP-binding protein probably involved in the active efflux of Acompounds. Nevertheless, there continues to exist a need in the art forpolynucleotides specific for Staphylococcus resistant to streptogramin Aand/or B and related compounds.

SUMMARY OF THE INVENTION

Accordingly, this invention aids in fulfilling this need in the art. Inparticular, this invention provides a purified peptide comprising anamino acid sequence selected from the group consisting of:

-   -   a) SEQ ID NO: 4 which corresponds to the complete amino acid        sequence of Vga B or fragments derived from SEQ ID NO: 4        containing at least 10 amino acids;    -   b) SEQ ID NO: 5 which corresponds to the complete amino acid        sequence of Vat C or fragments derived from SEQ ID NO: 5        containing at least 1.0 amino acids;    -   c) SEQ ID NO: 6 which corresponds to the complete amino acid        sequence of Vgb B or fragments derived from SEQ ID NO: 6        containing at least 10 amino acids;    -   d) SEQ ID NO: 7 which corresponds to a fragment of the amino        acid sequence of Vgb B;    -   e) SEQ ID NO: 8 which corresponds to a fragment of the amino        acid sequence of Vga B;    -   f) SEQ ID NO: 9 which corresponds to a fragment of the amino        acid sequence of Vat C; and    -   g) SEQ ID NO: 10 which corresponds to a fragment of the amino        acid sequence of Vat C.

This invention additionally provides a purified polynucleotidecomprising the nucleotide sequence selected from the group consistingof:

-   -   a) SEQ ID NO: 1 which corresponds to the complete nucleic acid        sequence of vga B or fragments derived from SEQ ID NO: 1        containing 15 to 40 nucleotides;    -   b) SEQ ID NO: 2 which corresponds to the complete nucleic acid        sequence of vat C or fragments derived from SEQ ID NO: 2        containing 15 to 40 nucleotides;    -   c) SEQ ID NO: 3 which corresponds to the complete nucleic acid        sequence of vgb B or fragments derived from SEQ ID NO: 3        containing 15 to 40 nucleotides;    -   d) SEQ ID NO: 11 which corresponds to the nucleic acid sequence        encoding the polypeptide of SEQ ID NO: 7;    -   e) SEQ ID NO: 12 which corresponds to the nucleic acid sequence        encoding the polypeptide of SEQ ID NO: 8;    -   f) SEQ ID NO: 13 which corresponds to the nucleic acid sequence        encoding the polypeptide of SEQ ID NO: 9; and    -   g) SEQ ID NO: 14 which corresponds to the nucleic acid sequence        encoding the polypeptide of SEQ ID NO: 10.

Furthermore, this invention includes a purified peptide comprising theamino acid sequence encoded by the nucleotide sequence selected from thegroup consisting of:

-   -   a) SEQ ID NO: 1,    -   b) SEQ ID NO: 2,    -   c) SEQ ID NO: 3,    -   d) SEQ ID NO: 11,    -   e) SEQ ID NO: 12,    -   f) SEQ ID NO: 13, and    -   g) SEQ ID NO: 14.

This invention also provides a composition comprising purifiedpolynucleotide sequences including at least one nucleotide sequence ofthe genes selected from the group consisting of polypeptides or genes orcDNA of vgaB, vatC, and vgbB, which are useful for the detection ofresistance to streptogramin A or to streptogramin B and relatedcompounds.

In another embodiment, this invention provides a composition ofpolynucleotide sequences encoding resistance to streptogramins andrelated compounds, or inducing this resistance in Gram-positivebacteria, wherein the composition comprises a combination of at leasttwo of the following nucleotide sequences: a) a nucleotide sequenceencoding an acetyltransferase conferring resistance to streptogramin Aand related compounds, b) a nucleotide sequence encoding a moleculecontaining ATP binding motifs conferring resistance to streptogramin Aand related compounds; and c) a nucleotide sequence encoding a lactonaseconferring resistance to streptogramin B and related compounds.

Furthermore, this invention provides a composition of polynucleotidesequences, wherein the sequence encoding a molecule containing ATPbinding motifs confers resistance to Staphylococci and particularly toS. aureus, and wherein the polynucleotide sequence corresponds to a vgaBnucleotide sequence represented by SEQ ID NO: 1 or a sequence having atleast 70% homology with vgaB complete nucleotide sequence, or to apolynucleotide hybridizing with SEQ ID NO: 1 under stringent conditions,or to a fragment containing between 20 and 30 nucleotides of SEQ ID NO:11 or SEQ ID NO: 12, or wherein the polynucleotide sequence encodes apolypeptide having at least 60% homology with the complete SEQ ID NO: 4or with SEQ ID NO: 7 or SEQ ID NO: 8.

Furthermore this invention relates to a composition of polynucleotidesequences, wherein the sequence encoding an acetyltransferase confersresistance to streptogramin A and related compounds in Staphylococci,and particularly in S. cohnii, and wherein the polynucleotide sequencecorresponds to a vatC nucleotide sequence represented by SEQ ID NO: 2 ora sequence having at least 70% homology with vatC complete nucleotidesequence, or to a polynucleotide hybridizing with SEQ ID NO: 2 understringent conditions, or to a fragment containing between 20 and 30nucleotides of SEQ ID NO: 13 or SEQ ID NO: 14, or wherein thepolynucleotide sequence encodes a polypeptide having at least 60%homology with the complete SEQ ID NO: 5 or with SEQ ID NO: 9 or SEQ IDNO: 10.

This invention also provides a composition of polynucleotide sequences,wherein the sequence encoding a lactonase confers resistance tostreptogramin B and related compounds in Staphylococci and particularlyin S. cohnii, and wherein the polynucleotide sequence corresponds to avgbB nucleotide sequence represented in SEQ ID NO: 3 or a sequencehaving at least 70% homology with vgbB complete nucleotide sequence, orto a polynucleotide hybridizing with SEQ ID NO: 3 under stringentconditions, or to a fragment containing between 20 and 40 nucleotides ofSEQ ID NO: 3, or wherein the polynucleotide sequence encodes apolypeptide having at least 60% homology with the complete SEQ ID NO: 6.

The invention also contemplates a composition of polynucleotidesequences, wherein at least a vatB nucleotide sequence encoding anacetyltransferase conferring resistance to streptogramin A and relatedcompounds is included in addition to a vgaB nucleotide sequence encodinga molecule containing ATP binding motifs conferring resistance tostreptogramin A.

Additionally, the invention includes a purified polynucleotide thathybridizes specifically under stringent conditions with a polynucleotidesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ IDNO: 14.

The invention further includes polynucleotide fragments comprising atleast 10 nucleotides capable of hybridization under stringent conditionswith any one of the nucleotide sequences enumerated above.

In another embodiment of the invention, a recombinant DNA sequencecomprising at least one nucleotide sequence enumerated above and underthe control of regulatory elements that regulate the expression ofresistance to antibiotics of the streptogramin family in a defined hostis provided.

Furthermore, the invention includes a recombinant vector comprising therecombinant DNA sequence noted above, wherein the vector comprises theplasmid pIP1633 or plasmid pIP1714.

The invention also includes a recombinant cell host comprising apolynucleotide sequence enumerated above or the recombinant vectordefined above.

In still a further embodiment of the invention, a method of detectingbacterial strains that contain the polynucleotide sequences set forthabove is provided.

Additionally, the invention includes kits for the detection of thepresence of bacterial strains that contain the polynucleotide sequencesset forth above.

The invention also contemplates antibodies recognizing peptide fragmentsor polypeptides encoded by the polynucleotide sequences enumeratedabove.

Still further, the invention provides for a screening method for activeantibiotics and/or molecules for the treatment of infections due toGram-positive bacteria, particularly staphylococci, based on thedetection of activity of these antibiotics and/or molecules on bacteriahaving the resistance phenotype to streptogramins.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be more fully described with reference to thedrawings in which:

FIGS. 1A and 1B are the restriction maps of the 5.5 kb BglII fragmentand of the 2.4 kb HindIII-HaeIII fragment of pIP1633, respectively. Bothfragments confer resistance to streptogramin A and related compounds.The strategy for sequencing the 2.4 kb HindIII-HaeIII fragment is givenin FIG. 1B. Restriction enzyme abbreviations: Ba, BamHI; Bg, BgIII; E,EcoRI; H, HindIII; X, XbaI.

FIG. 2 is the nucleotide sequence and deduced amino acid sequence of2411 nucleotides from pIP1633, which contains the gene vgaB of S. aureusconferring resistance to streptogramin A and related compounds. Theputative ribosome binding site (RBS) is underlined. The amino acids arealigned with the second nucleotide of each codon. Asterisks indicate thein-frame stop codons. The A and B ATP-binding motifs described by Walkeret al. (1982) and detected within each of the two ATP-domains are boxed.The conserved motif SGG of the two copies of loop 3 described by Hyde etal. (1990) is underlined. Relevant restriction sites are shown.

FIG. 3 is the amino acid sequence alignment of the predicted 60 and 61kDa proteins encoded by Vga (Allignet et al., 1992, accession No:m90056) and VgaB (FIG. 2), respectively. Identical residues areindicated by asterisks and conservative changes are shown by singledots. The A and B motifs of Walker et al. (1982) are in bold type (WA,WB). The conserved motif SGG of the two copies of loop 3 described byHyde et al. (1990) is underlined.

FIG. 4 is a restriction map of the plasmid pIP1714 carrying the genesvatC and vgbB as well as the genes pre and repB of S. cohnii strainBM10711 resistant to the synergistic mixtures of streptogramins A and B.

FIG. 5 is the nucleotide sequence and deduced amino acid sequence of1727 nucleotide from pIP1714, which contains the gene vgbB and vatC ofS. cohnii. Relevant restriction sites are shown.

FIGS. 6A, 6B, and 6C represent oligonucleotide primers for hybridizationunder stringent conditions with vatC, vgbB, and vgaB respectively.

FIG. 7 represents SEQ ID NOs: 1-14.

DETAILED DESCRIPTION OF THE INVENTION

It has now been determined that bacteria from the Staphylococcus genuscarry a vgaB gene, which encodes a putative ATP-binding protein thatconfers resistance to streptogramin A and structurally similarcompounds. It has also now been determined that bacteria from theStaphylococcus genus carry a vgbB gene, which encodes a lactonase thatconfers resistance to streptogramin B and structurally similarcompounds, and a vatC gene, which encodes an acetyltransferase thatconfers resistance to streptogramin A and structurally similarcompounds.

Novel polynucleotides corresponding to the vgaB, vgbB, and vatC genesfrom various strains of Staphylococcus have been isolated and sequenced,and it has been surprisingly demonstrated that these new polynucleotidesmake it possible to design oligonucleotide probes or primers. Thesepolynucleotides include the following:

-   -   a) SEQ ID NO: 1,    -   b) SEQ ID NO: 2,    -   c) SEQ ID NO: 3,    -   d) SEQ ID NO: 11,    -   e) SEQ ID NO: 12,    -   f) SEQ ID NO: 13, and    -   g) SEQ ID NO: 14.

This invention provides specific pairs of oligonucleotide primers orprobes that hybridize specifically, under stringent hybridizationconditions as defined hereinafter, to the nucleic acid (RNA or DNA) froma particular strain of the Staphylococcus genus. These oligonucleotideprimers include the following: a) Oligo I5′-AAGTCGACTGACAATATGAGTGGTGG-3′ Oligo II5′-CTGCAGATGCCTCAACAGCATCGATATCC-3′ b) Oligo III5′-ATGAATTCGCAAATCAGCAAGG-3′ Oligo IV 5′-TCGTCTCGAGCTCTAGGTCC-3′ c)Oligo V 5′-CAGCAGTCTAGATCAGAGTGG-3′ Oligo VI 5′-CATACGGATCCACCTTTTCC-3′.

In a specific embodiment of the present invention, the purifiedpolynucleotides useful for detecting Staphylococcal strains can be usedin combination in order to detect bacteria belonging to Staphylococci ina biological sample. Thus, the present invention also provides detectionmethods and kits comprising combinations of the purified polynucleotidesaccording to the invention. The purified oligonucleotides of theinvention are also useful as primers for use in amplification reactionsor as nucleic acid probes.

By “polynucleotides” according to the invention is meant the sequencesreferred to as SEQ ID NOs: 1, 2, 3, OR 11, 12, 13, 14 and thecomplementary sequences and/or the sequences of polynucleotides whichhybridize to the referred sequences in high stringent conditions andwhich are used for detecting staphylococcal strains carrying a geneencoding resistance to streptogramin A or to streptogramin B.

By “active molecule” according to the invention is meant a moleculecapable of inhibiting the activity of the purified polypeptide asdefined in the present invention or capable of inhibiting the bacterialculture of staphylococcal strains.

Thus, the polynucleotides of SEQ ID NOs: 1-3 and 11-14 and theirfragments can be used to select nucleotide primers notably for anamplification reaction, such as the amplification reactions furtherdescribed.

PCR is described in the U.S. Pat. No. 4,683,202 granted to Cetus Corp.The amplified fragments may be identified by agarose or polyacrylamidegel electrophoresis, or by a capillary electrophoresis, or alternativelyby a chromatography technique (gel filtration, hydrophobicchromatography, or ion exchange chromatography). The specificity of theamplification can be ensured by a molecular hybridization using asnucleic probes the polynucleotides of SEQ ID NOs: 1-3 and 11-14 andtheir fragments, oligonucleotides that are complementary to thesepolynucleotides or fragments thereof, or their amplification productsthemselves.

Amplified nucleotide fragments are useful as probes in hybridizationreactions in order to detect the presence of one polynucleotideaccording to the present invention or in order to detect the presence ofa bacteria of Staphylococcal strain carrying genes encoding resistanceto streptogramin A or streptogramin B, in a biological sample. Thisinvention also provides the amplified nucleic acid fragments(“amplicons”) defined herein above. These probes and amplicons can beradioactively or non-radioactively labeled, using for example enzymes orfluorescent compounds.

Preferred nucleic acid fragments that can serve as primers according tothe present invention are the following:

-   -   polynucleotides of sequence SEQ ID NOs: 1-3 and 11-14; and    -   polynucleotides having a length from 20 to 30 consecutive        nucleotides from a polynucleotide selected from the group        consisting of polynucleotides of sequences SEQ ID NO: 11 to SEQ        ID NO: 14 or from 20 to 40 consecutive nucleotides from a        polynucleotide of SEQ ID NO: 3        The primers can also be used as oligonucleotide probes to        specifically detect a polynucleotide according to the invention.

Other techniques related to nucleic acid amplification can also be usedand are generally preferred to the PCR technique.

The Strand Displacement Amplification (SDA) technique (Walker et al.,1992) is an isothermal amplification technique based on the ability of arestriction enzyme to cleave one of the strands at a recognition site(which is under a hemiphosphorothioate form), and on the property of aDNA polymerase to initiate the synthesis of a new strand from the 3′ OHend generated by the restriction enzyme and on the property of this DNApolymerase to displace the previously synthesized strand being localizeddownstream.

The SDA amplification technique is more easily performed than PCR (asingle thermostated water bath device is necessary), and is faster thanthe other amplification methods. Thus, the present invention alsocomprises using the nucleic acid fragments according to the invention(primers) in a method of DNA or RNA amplification according to the SDAtechnique. The polynucleotides of SEQ ID NOs: 1-3 and 11-14 and theirfragments, especially the primers according to the invention, are usefulas technical means for performing different target nucleic acidamplification methods such as:

-   -   TAS (Transcription-based Amplification System), described by        Kwoh et al. in 1989;    -   SR (Self-Sustained Sequence Replication), described by Guatelli        et al. in 1990;    -   NASBA (Nucleic acid Sequence Based Amplification), described by        Kievitis et al. in 1991; and    -   TMA (Transcription Mediated Amplification).

The polynucleotides of SEQ ID NOs: 1-3 and 11-14 and their fragments,especially the primers according to the invention, are also useful astechnical means for performing methods for amplification or modificationof a nucleic acid used as a probe, such as:

-   -   LCR (Ligase Chain Reaction), described by Landegren et al. in        1988 and improved by Barany et al. in 1991, who employ a        thermostable ligase;    -   RCR (Repair Chain Reaction), described by Segev et al. in 1992;    -   CPR (Cycling Probe Reaction), described by Duck et al. in 1990;        and    -   Q-beta replicase reaction, described by Miele et al. in 1983 and        improved by Chu et al. in 1986, Lizardi et al. in 1988, and by        Burg et al. and Stone et al. in 1996.

When the target polynucleotide to be detected is RNA, for example mRNA,a reverse transcriptase enzyme can be used before the amplificationreaction in order to obtain a cDNA from the RNA contained in thebiological sample. The generated cDNA can be subsequently used as thenucleic acid target for the primers or the probes used in anamplification process or a detection process according to the presentinvention.

Nucleic probes according to the present invention are specific to detecta polynucleotide of the invention. By “specific probes” according to theinvention is meant any oligonucleotide that hybridizes with onepolynucleotide of SEQ ID NOs: 1-3 and 11-14 and which does not hybridizewith unrelated sequences. Preferred oligonucleotide probes according tothe invention are oligonucleotides I-VI.

In a specific embodiment, the purified polynucleotides according to thepresent invention encompass polynucleotides having at least 80% homologyin their nucleic acid sequences with polynucleotides of SEQ ID NO: 11 toSEQ ID NO: 14, at least 70% identity with SEQ ID NO: 1 to 3. Bypercentage of nucleotide homology according to the present invention isintended a percentage of identity between the corresponding bases of twohomologous polynucleotides, this percentage of identity being purelystatistical and the differences between two homologous polynucleotidesbeing located at random and on the whole length of said polynucleotides.

The oligonucleotide probes according to the present invention hybridizespecifically with a DNA or RNA molecule comprising all or part of onepolynucleotide among SEQ ID NOs: 1-3 and 11-14 under stringentconditions. As an illustrative embodiment, the stringent hybridizationconditions used in order to specifically detect a polynucleotideaccording to the present invention are advantageously the following:

Prehybridization and hybridization are performed at 68° C. in a mixturecontaining:

-   -   5× SSPE (1× SSPE is 0.3 M NaCl, 30 mM tri-sodium citrate    -   5× Denhardt's solution    -   0.5% (w/v) sodium dodecyl sulfate (SDS); and    -   100 μg ml⁻¹ salmon sperm DNA        The washings are performed as follows:    -   Two washings at laboratory temperature for 10 min. in the        presence of 2× SSPE and 0.1% SDS;    -   One washing at 68° C. for 15 min. in the presence of 1× SSPE,        0.1% SDS; and    -   One washing at 68° C. for 15 min. in the presence of 0.1× SSPE        and 0.1% SDS.

The non-labeled polynucleotides or oligonucleotides of the invention canbe directly used as probes. Nevertheless, the polynucleotides oroligonucleotides are generally labeled with a radioactive element (³²P,³⁵S, ³H, ¹²⁵I) or by a non-isotopic molecule (for example, biotin,acetylaminofluorene, digoxigenin, 5-bromodesoxyuridin, fluorescein) inorder to generate probes that are useful for numerous applications.Examples of non-radioactive labeling of nucleic acid fragments aredescribed in the French Patent No. FR 78 10975 or by Urdea et al. orSanchez-Pescador et al. 1988.

Other labeling techniques can also be used, such as those described inthe French patents 2 422 956 and 2 518 755. The hybridization step maybe performed in different ways (Matthews et al. 1988). A general methodcomprises immobilizing the nucleic acid that has been extracted from thebiological sample on a substrate (nitrocellulose, nylon, polystyrene)and then incubating, in defined conditions, the target nucleic acid withthe probe. Subsequent to the hybridization step, the excess amount ofthe specific probe is discarded, and the hybrid molecules formed aredetected by an appropriate method (radioactivity, fluorescence, orenzyme activity measurement).

Advantageously, the probes according to the present invention can havestructural characteristics such that they allow signal amplification,such structural characteristics being, for example, branched DNA probesas those described by Urdea et al. in 1991 or in the European Patent No.0 225 807 (Chiron).

In another advantageous embodiment of the present invention, the probesdescribed herein can be used as “capture probes”, and are for thispurpose immobilized on a substrate in order to capture the targetnucleic acid contained in a biological sample. The captured targetnucleic acid is subsequently detected with a second probe, whichrecognizes a sequence of the target nucleic acid that is different fromthe sequence recognized by the capture probe.

The oligonucleotide fragments useful as probes or primers according tothe present invention can be prepared by cleavage of the polynucleotidesof SEQ ID NOs: 1-3 and 11-14 by restriction enzymes, as described inSambrook et al. in 1989. Another appropriate preparation process of thenucleic acids of the invention containing at most 200 nucleotides (or200 bp if these molecules are double-stranded) comprises the followingsteps:

-   -   synthesizing DNA using the automated method of        beta-cyanethylphosphoramidite described in 1986;    -   cloning the thus obtained nucleic acids in an appropriate        vector; and    -   purifying the nucleic acid by hybridizing to an appropriate        probe according to the present invention.

A chemical method for producing the nucleic acids according to theinvention, which have a length of more than 200 nucleotides (or 200 bpif these molecules are double-stranded), comprises the following steps:

-   -   Assembling the chemically synthesized oligonucleotides having        different restriction sites at each end;    -   cloning the thus obtained nucleic acids in an appropriate        vector; and    -   purifying the nucleic acid by hybridizing to an appropriate        probe according to the present invention.

The oligonucleotide probes according to the present invention can alsobe used in a detection device comprising a matrix library of probesimmobilized on a substrate, the sequence of each probe of a given lengthbeing localized in a shift of one or several bases, one from the other,each probe of the matrix library thus being complementary to a distinctsequence of the target nucleic acid. Optionally, the substrate of thematrix can be a material able to act as an electron donor, the detectionof the matrix positions in which hybridization has occurred beingsubsequently determined by an electronic device. Such matrix librariesof probes and methods of specific detection of a target nucleic acid aredescribed in the European patent application No. 0 713 016, or PCTApplication No. WO 95 33846, or also PCT Application No. WO 95 11995(Affymax Technologies), PCT Application No. WO 97 02357 (AffymetrixInc.), and also in U.S. Pat. No. 5,202,231 (Drmanac), said patents andpatent applications being herein incorporated by reference.

The present invention also pertains to a family of recombinant plasmidscontaining at least a nucleic acid according to the invention. Accordingto an advantageous embodiment, a recombinant plasmid comprises apolynucleotide of SEQ ID NOs: 1-3 and 11-14 or one nucleic fragmentthereof. More specifically, the following plasmids are part of theinvention: pIP1633 and pIP1714.

The present invention is also directed to the full length codingsequences of the vgaB, vgbB, and vatC genes from Staphylococci that areavailable using the purified polynucleotides according to the presentinvention, as well as to the polypeptide enzymes encoded by these fulllength coding sequences. In a specific embodiment of the presentinvention, the full length coding sequences of the vgaB, vgbB, and vatCgenes are isolated from a plasmid or cosmid library of the genome ofStaphylococci that have been screened with the oligonucleotide probesaccording to the present invention. The selected positive plasmid orcosmid clones hybridizing with the oligonucleotide probes of theinvention are then sequenced in order to characterize the correspondingfull length coding sequence, and the DNA insert of interest is thencloned in an expression vector in order to produce the corresponding ATPbinding motif conferring resistance to streptogramin A and relatedcompounds, acetyltransferase conferring resistance to streptogramin Aand related compounds, or lactonase conferring resistance tostreptogramin B and related compounds.

A suitable vector for the expression in bacteria and in particular in E.coli, is the pQE-30 vector (QIAexpress) that allows the production of arecombinant protein containing a 6×His affinity tag. The 6×His tag isplaced at the C-terminus of the recombinant polypeptide ATP bindingmotif conferring resistance to streptogramin A and related compounds,acetyltransferase conferring resistance to streptogramin A and relatedcompounds or lactonase conferring resistance to streptogramin B andrelated compounds, which allows a subsequent efficient purification ofthe recombinant polypeptide ATP binding motif conferring resistance tostreptogramin A and related compounds, acetyltransferase conferringresistance to streptogramin A and related compounds, or lactonaseconferring resistance to streptogramin B and related compounds bypassage onto a nickel or copper affinity chromatography column. Thenickel chromatography column can contain the Ni-NTA resin (Porath et al.1975).

The polypeptides according to the invention can also be prepared byconventional methods of chemical synthesis, either in a homogenoussolution or in solid phase. As an illustrative embodiment of suchchemical polypeptide synthesis techniques the homogenous solutiontechnique described by Houbenweyl in 1974 may be cited.

The polypeptides according to the invention can be characterized bybinding onto an immunoaffinity chromatography column on which polyclonalor monoclonal antibodies directed to a polypeptide among the ATP bindingmotif conferring resistance to streptogramin A and related compounds,acetyltransferase conferring resistance to streptogramin A and relatedcompounds, or lactonase conferring resistance to streptogramin B andrelated compounds of the invention have previously been immobilized.

Another object of the present invention comprises a polypeptide producedby the genetic engineering techniques or a polypeptide synthesizedchemically as above described.

The polypeptide ATP binding motif conferring resistance to streptograminA and related compounds, acetyltransferase conferring resistance tostreptogramin A and related compounds, or lactonase conferringresistance to streptogramin B and related compounds according to thepresent invention are useful for the preparation of polyclonal ormonoclonal antibodies that recognize the polypeptides or fragmentsthereof. The monoclonal antibodies can be prepared from hybridomasaccording to the technique described by Kohler and Milstein in 1975. Thepolyclonal antibodies can be prepared by immunization of a mammal,especially a mouse or a rabbit, with a polypeptide according to theinvention that is combined with an adjuvant, and then by purifyingspecific antibodies contained in the serum of the immunized animal on aaffinity chromatography column on which has previously been immobilizedthe polypeptide that has been used as the antigen.

Consequently, the invention is also directed to a method for detectingspecifically the presence of a polypeptide according to the invention ina biological sample. The method comprises:

-   -   a) bringing into contact the biological sample with an antibody        according to the invention; and    -   b) detecting antigen-antibody complex formed.

Also part of the invention is a diagnostic kit for in vitro detectingthe presence of a polypeptide according to the present invention in abiological sample. The kit comprises:

-   -   a polyclonal or monoclonal antibody as described above,        optionally labeled; and    -   a reagent allowing the detection of the antigen-antibody        complexes formed, wherein the reagent carries optionally a        label, or being able to be recognized itself by a labeled        reagent, more particularly in the case when the above-mentioned        monoclonal or polyclonal antibody is not labeled by itself.

Indeed, the monoclonal or polyclonal antibodies according to the presentinvention are useful as detection means in order to identify orcharacterize a Staphylococcal strain carrying genes encoding resistanceto streptogramin A or streptogramin B.

The invention also pertains to:

A purified polypeptide or a peptide fragment having at least 10 aminoacids, which is recognized by antibodies directed against apolynucleotide sequence conferring resistance to streptogramin andrelated compounds, corresponding to a polynucleotide sequence accordingto the invention.

A polynucleotide comprising the full length coding sequence of aStaphylococcus streptogramin A and/or B resistant gene containing apolynucleotide sequence according to the invention.

A monoclonal or polyclonal antibody directed against a polypeptide or apeptide fragment encoded by the polynucleotide sequences according tothe invention.

A method of detecting the presence of bacterium harboring thepolynucleotide sequences according to the invention in a biologicalsample comprising:

-   -   a) contacting bacterial DNA of the biological sample with a        primer or a probe according to the invention, which hybridizes        with a nucleotide sequence encoding resistance to        streptogramins;    -   b) amplifying the nucleotide sequence using said primer or said        probe; and    -   c) detecting the hybridized complex formed between said primer        or probe with the DNA.

A kit for detecting the presence of bacterium having resistance tostreptogramin A and/or streptogramin B and harboring the polynucleotidesequences according to the invention in a biological sample, said kitcomprising:

-   -   a) a polynucleotide probe according to the invention; and    -   b) reagents necessary to perform a nucleic acid hybridization        reaction.

A kit for detecting the presence of bacterium having resistance tostreptogramin A and harboring the polynucleotide sequences according tothe invention in a biological sample, said kit comprising:

-   -   a) a polynucleotide probe according to the invention; and    -   b) reagents necessary to perform a nucleic acid hybridization        reaction.

A method of screening active antibiotics for the treatment of theinfections due to Gram-positive bacteria, comprising the steps of:

-   -   a) bringing into contact a Gram-positive bacteria having a        resistance to streptogramin A or streptogramin B and related        compounds and containing the polynucleotide sequences according        to the invention with the antibiotic; and    -   b) measuring an activity of the antibiotic on the bacteria        having a resistance to streptogramins and related compounds.

A method of screening for active synthetic molecules capable ofpenetrating into a bacteria of the family of staphylococci, wherein theinhibiting activity of these molecules is tested on at least apolypeptide encoded by the polynucleotide sequences according to theinvention comprising the steps of:

-   -   a) contacting a sample of said active molecules with the        bacteria;    -   b) testing the capacity of the active molecules to penetrate        into the bacteria and the capacity of inhibiting a bacterial        culture at various concentration of the molecules; and    -   c) choosing the active molecule that provides an inhibitory        effect of at least 80% on the bacterial culture compared to an        untreated culture.

An in vitro method of screening for active molecules capable ofinhibiting a polypeptide encoded by the polynucleotide sequencesaccording to the invention, wherein the inhibiting activity of thesemolecules is tested on at least said polypeptide, said method comprisingthe steps of:

-   -   a) extracting a purified polypeptide according to the invention;    -   b) contacting the active molecules with said purified        polypeptide;    -   c) testing the capacity of the active molecules, at various        concentrations, to inhibit the activity of the purified        polypeptide; and    -   d) choosing the active molecule that provides an inhibitory        effect of at least 80% on the activity of the said purified        polypeptide.

A composition of a polynucleotide sequence encoding resistance tostreptogramins and related compounds, or inducing resistance inGram-positive bacteria, wherein said composition comprises a nucleotidesequence corresponding to the resistance phenotype of the plasmidpIP1633 deposited with the C.N.C.M. under the Accession No. I-1768 andof the plasmid pIP1680 deposited with the C.N.C.M. under the AccessionNo. I-1767 and of the plasmid pIP1714 deposited with the C.N.C.M. underthe number I-1877 on Jun. 18, 1997.

A method of detecting the presence of bacterium harboring thepolynucleotide sequences according to the invention in a biologicalsample, said method comprising the steps of:

-   -   a) contacting said sample with an antibody according to the        invention that recognizes a polypeptide encoded by said        polynucleotide sequences; and    -   b) detecting said complex.

A diagnostic kit for in vitro detecting the presence of bacteriumharboring the polynucleotide sequences according to the invention in abiological sample, said kit comprising:

-   -   a) a predetermined quantity of monoclonal or polyclonal        antibodies according to the invention;    -   b) reagents necessary to perform an immunological reaction        between the antibodies and a polypeptide encoded by said        polynucleotide sequences; and    -   c) reagents necessary for detecting said complex between the        antibodies and the polypeptide encoded by said polynucleotide        sequences.

The inhibiting activity of the molecules can be readily evaluated by oneskilled in the art. For example, the inhibiting activity of Vga B can betested by detecting its ATP hydrolysis as described in J. I. Ross et al.(1990), Mol. Microbiol. 4(7):1207-1214 regarding the rate evaluation ofthe active efflux of antibiotics from a cell. Ross et al. use adifferent gene, but their gene product functions as a drug efflux pumpin the same way as Vga B does.

The inhibiting activity of Vat C can be tested by visualizing theacetylation reaction as described in Allignet et al. (1993) regardingthe mechanism of inactivation of A-type compounds conferred by plasmidspIP680 and pIP1156 by thick layer chromatography and NMR.

The inhibiting activity of Vgb B can be tested by detecting thedegradation of streptogramin B or a related compound by amicrobiological test as described in Allignet et al. (1988).

Plasmids containing the polynucleotides from Staphylococci, which conferstreptogramin A and/or B resistance, are referred to herein by thefollowing accession numbers: Plasmid Accession No. pIP1714 I-1877pIP1633 I-1768 pIP680 I-1767and they have been inserted into vectors which have been deposited atthe Collection Nationale de Cultures de Microorganismes (“C.N.C.M.”)Institut Pasteur, 28, rue du Docteur Roux, F-75724 Paris Cedex 15,France on Jun. 18, 1997, and Aug. 7, 1996, respectively.

EXAMPLES Example 1

Cloning of the vgaB gene carried by plasmid pIP1633 pIP1633 was isolatedfrom a S. aureus transconjugant strain, BM12235, obtained from the donorwild-type S. aureus strain, BM3385 (Allignet and El Solh, 1995). Thisplasmid carried the vatB gene located on a 5.5 BglII fragment, but theother described streptogramin A resistant (SgA^(r)) genes were notdetected either by hybridization experiments or by PCR (Allignet and EISolh, 1995). Since the gene vga was carried by all the testedstaphylococcal plasmids containing the vat gene (Allignet et al., 1996),the presence of a vga-related gene was suspected in pIP1633. Wetherefore searched this gene in the recombinant plasmid, pIP1675 (FIG.1A), containing the vatB-5.5 BglII fragment of pIP1633.

First, the 2.4 kb HindIII-Haelll fragment of pIP1675, which containsonly 10 nucleotide from vatB, was inserted into plasmid pOX300, and therecombinant plasmid, pIP1717 (FIG. 1B), was introduced byelectroporation into the S. aureus recipient, RN4220 (Kreiswirth et al.,1983). Plasmid pOX300, also named pOX7, (Dyke and Curnock, 1989), is ahybrid of pUC18 and pE194ts and replicates in E. coli where it confersresistance to ampicillin and to erythromycin, and in S. aureus whereonly resistance to erythromycin is expressed. The S. aureustransformants selected on 10 μg/ml erythromycin were resistant tostreptogramin A and related compounds (PIIA MICs=8-16 μg/ml). Thus, the2.4-kb HindIII-HaeIII insert of pIP1717 (FIG. 1B) probably carried astreptogramin A resistance gene and was sequenced. The nucleotide(nucleotide) sequence of this fragment was determined by the dideoxymethod (Sanger et al., 1977) with the reagents and the procedurerecommended by the suppliers of the T⁷ sequencing kit (PharmaciaInternational). Arrows indicate the direction and extent of eachdideoxy-sequencing reaction. (FIG. 1B).

Example 2 The Nucleotide Sequence of the vgaB Gene

The strategy of sequencing on both strands is outlined in FIG. 1 and thesequence of the 2411-bp HindIII-HaeIII insert is given in FIG. 2. Anopen reading frame (ORF) of 1674 nucleotide extending from nucleotide682 to 2356 was detected on the same strand as vatB (FIG. 2). The 1674nucleotide ORF contained an ATG start codon at nucleotide 700 to 702 andwas preceded by an 8 nucleotide putative RBS. The ΔG (free energy ofassociation) of interaction of the most stable structure between thisputative RBS and the 3′-terminus of the 165 rRNA (MacLaughlin et al.,1981; Moran et al., 1982) calculated according to Tinoco et al. (1973)was −79.4 kJ/mol. The sequence located between the ATG codon and the TAAstop codon at nucleotide 2356 to 2358 may encode a 552 amino acidprotein of 61,327 daltons (Da). This putative gene, named vgaB, had58.8% nucleotide identity with the 1572 bp gene, vga (Allignet et al.,1992). The G+C content of vgaB (27.2%) is similar to that of vga (29%),but both values are slightly lower than those of the staphylococcalgenome (32 to 36%) (KIoos and Schleifer, 1986). The nucleotide sequenceof vgaB has been submitted to the GenBank/EMBL data bank under accessionno. u82085.

Example 3 Amino Acid Sequence Analysis of VgaB

The predicted translation product of the vgaB gene, VgaB, has acalculated isoelectric point (pI) of 9.60. The hydropathy plot of theVgaB sequence according to the algorithm of Kyte and Doolittle (1982)indicates the protein to be hydrophilic. No similarity to known signalsequences of secreted proteins (von Heijne, 1986; Watson, 1984) wasobserved.

The amino acid sequence of VgaB was compared with the sequencesavailable in databases (GenBank, release 97.0; EMBL, release 48;SwissProt, release 34). Significant similarity to the ATP-bindingdomains of numerous ATP-binding Cassette (ABC) proteins was found. Theprotein giving the best match was Vga (48.3% identical amino acid, 70.4%similar amino acid). VgaB and Vga each contain two ATP-binding domainssharing 38.8% and 39.1% identical amino acid, respectively. Each ofthese domains includes the two ATP-binding motifs described by Walker etal. (1982) (FIG. 2). Moreover, the highly conserved SGG sequence of loop3 found between the two ATP-binding motifs of all investigatedATP-binding proteins (Barrasa et al., 1995; Hyde et al., 1990) wasdetected in Vga (Allignet et al., 1992) and VgaB (FIG. 2). According tothe predicted tertiary structure of ABC model cassette, this loop wouldbe conveniently located to interact with the cell membrane (Hyde et al.,1990). The inter-ATP-binding domain of VgaB is more rich in glutamine(11 Q in 155 amino acid total) than the rest of the sequence of theprotein (11 Q/397 amino acid). In contrast, the proportion of glutaminein the inter-ATP-binding domain of Vga is similar to that in the otherpart of the protein (4 Q/156 amino acid and 14 Q/366 amino acid,respectively). Neither Vga nor VgaB contains hydrophobic transmembranedomains.

The ABC protein MsrA (Ross et al., 1990) is the most similar to Vga andVgaB (35.2% and 34.4% identical amino acid, respectively). MsrA confersresistance to erythromycin by increasing the efflux of this antibioticand to streptogramin B by a mechanism not yet elucidated. MsrA containstwo ATP-binding domains with 31.8% amino acid identity and separated bya Q-linker, but no hydrophobic stretches that might be potentialmembrane spanning domains. The hydrophobic proteins, which are expectedto interact with MsrA, are those encoded by similar genes mapping nearMsrA in two staphylococcal strains (smpA, smpB) and also those on thechromosome of the S. aureus recipient strain, RN4220 (smpC), which doesnot carry msrA (Ross et al., 1995). Ross et al. (1996) have recentlyreported that SmpC found in the chromosome of RN4220 is not essentialfor the expression of resistance to erythromycin conferred by MsrA.Thus, further experiments are required to elucidate the mechanisms ofresistance conferred by msrA, vga, or vgaB genes.

Several ABC transporters, which do not have alternating hydrophobicdomains, have been grouped in a subfamily in order to distinguish themfrom the members of the ABC₂ transporter subfamily, the members of whichcontain hydrophobic transmembrane domains (Barrasa et al., 1995; Olanoet al., 1995; Peschke et al., 1995). Thus, VgaB may be considered as anew member of the former ABC transporter subfamily. Excluding VgaB, Vga,and MsrA, most of the known ABC transporters that contain twoATP-binding cassettes but no hydrophobic domain(s) were found inantibiotic or antibiotic producing microorganisms in which they areinvolved in the active excretion of these molecules. These transportersare encoded by the following genes: ardl, an amino-acylnucleosideantibiotic resistance gene from Streptomyces capreolus (Barrasa et al.,1995); carA, a carbomycin-resistance gene from Streptomycesthermotolerans (Schoner et al., 1992); ImrC, a lincomycin-resistancegene from Streptomyces lincolnensis (Peschke et al., 1995); oleB, anoleandomycin-resistance gene from Streptomyces antibioticus (Olano etal., 1995); srmB, a spiramycin-resistance gene from Streptomycesambofaciens (Geistlich et al., 1992); tlrC, a tylosin-resistance genefrom Streptomyces fradiae (Rosteck et al., 1991); and petT, a pep5epidermin-resistance gene from Staphylococcus epidermidis (Meyer et al.,1995). The amino acid identity between each of these latter ABCtransporters and VgaB is between 23.6% and 28.7%.

Degenerate primers designed from an analysis of the alignment of theamino acid sequence of Vga and VgaB may be helpful to detect suchputative genes by PCR experiments. In the streptogramins producers, thedescribed resistance to these antibiotics consists of streptogramin Ainactivation by an as yet unknown mechanism (Fierro et al., 1989),streptogramin B inactivation by a lactonase (Kim et al., 1974) andputative increased export of streptogramin A and streptogramin B by anintegral membrane protein, Ptr, exploiting transmembrane protongradients (Blanc et al., 1995). The NMR spectra of the modified Acompounds may be analyzed to verify if their inactivation in theantibiotic producers is similar to that due to the proteins Vat or VatB,which transfer an o-acetyl group to position C14 of PIIA (Allignet etal., 1993). Interestingly, the staphylococcal gene vgb (Allignet et al.,1988) found in most plasmids carrying vga and vat (Allignet et al.,1996), encodes a protein inactivating streptogramin B and relatedcompounds by cleavage of the lactone ring.

Example 4 Distribution and Location of the vgaB Gene in 52 SgA^(R) andIndependent Wild-Type Staphylococci

A recombinant plasmid containing a fragment of vgaB, pIP1705, wasconstructed to serve as a probe in hybridization experiments understringent conditions as described previously (Allignet et al., 1996).pIP1705 consists of pUC19 cleaved with SalI and PstI, and an insert of1051 bp amplified from within vgaB by the following primers, whichintroduce PstI or SalI sites: Oligo I 5′-AAGTCGACTGACAATATGAGTGGTGG-3′       SalI Oligo II 5′-CTGCAGATGCCTCAACAGCATCGATATCC-3′           Pstl

The 52 SgA^(r) staphylococci investigated (Allignet et al., 1996; ElSolh et al., 1980; Loncle et al., 1993) included 10 strains (7S. aureus,1S. simulans, 1S. haemolyticus, and 1S. cohnii urealyticum), whichharbored 26 to 45 kb plasmids containing vga, vat, and vgb; 21 strains(20 S. aureus and one S. epidermidis), which harbored 50 to 90 kbplasmids containing vatB; 16 strains (12 S. epidermidis, three S.haemolyticus and one S. aureus) with 6 to 15 kb plasmids containing vga;one S. epidermidis strain which harbored a plasmid of approximately 20kb containing vga-vat; and four S. aureus strains, which do not carrynucleotide sequences hybridizing with vat, vatB, vga, or vgb. Nucleotidesequences hybridizing with pIP1705 were found only in the 21 largeplasmids containing vatB. In all these 21 plasmids including pIP1633,the hybridizing nucleotide sequences were detected on a 1.5 kb EcoRIfragment, which also hybridized with vatB, suggesting that vgaB and vatBhave conserved relative positions.

Example 5 Results Concerning vatC and vgbB Genes

The Staphylococcus cohnii strain, BM10711, resistant to the synergisticmixtures streptogramin A and streptogramin B and related compounds(pristinamycin, virginiamycin, synergistin, mikamycin,Quinupristin-Dalfopristin) was analyzed. This strain was isolated atDouera hospital (Algeria) where the pristinamycin was frequently usedtopically. The strain was isolated (Liassin et al., 1997) from a sampleprovided from a cupboard located in a room occupied by patientssuffering from chronic osteomyelitis.

The strain BM10711 harbored several plasmids including pIP1714 (5 kb).This plasmid was isolated by electroporation in a S. aureus recipientstrain, RN4220. The transformant, harboring pIP1714, was selected onBHIA containing 10 μg/ml pristinamycin IIA. Plasmid pIP1714 conferredresistances to streptogramin A and streptogramin B and relatedcompounds.

Plasmid pIP1714 was linearized by cleavage with HindIII and cloned inthe HindIII site of the vector pOX7 also named pOX300 (Dyke et al.,1989, FEMS Microbiol. Lett. 58:209-216). pOX7 results from thecointegration of the E. coli vector, pUC18, and S. aureus plasmid,pE194. The recombinant plasmid pIP1715 consisting of pOX7 and pIP1714was used to sequence pIP1714 in its entirety. The gene vatC (636nucleotides) encoding an acetyltransferase inactivating streptogramin Aand related compounds and the gene vgbB (885 nucleotides) encoding alactonase inactivating streptogramin B and related compounds were foundto be carried by this plasmid. The gene vatC had 71.7, 62.2 and 64.1%nucleotides identity with vat-related gene, vatB and satA respectivelyand the gene vgbB presents 69.5% nucleotides identity with the gene vgb.

VatC acetyltransferase exhibits significant similarity withacetyltransferases having the same enzymatic activity and encoded by thegenes vatC, vatB, and sat (respectively 69.8, 58.2 and 66.0% amino acidsidentity). These proteins belong to a family of xenobioticacetyltransferases modifying various substrates including streptograminA and related antibiotics. VgbB lactonase exhibits as well significantsimilarity with Vgb inactivating streptogramin B and related (67.0%amino acids identity).

The two other genes carried by pIP1714 are pre and repB, encodingproteins involved in mobilization and replication, respectively. Thesetwo genes are homologous to those carried by the staphylococcal plasmid,pUB110 (McKenzie et al., 1986, Plasmid 15:93-103). Moreover, as reportedin FIG. 5, the intergenic sequences of pIP1714 delimited by vatC andrepB also exhibited significant similarities with pUB110.

Example 6 Plasmid DNA Isolation from PIIA^(R) Staphylococci

The staphylococci were grown after overnight incubation at 37° C. in 200ml BHI containing 10 μg/ml of PIIA. After 15 min centrifugation at 8000rpm, the pellet was resuspended in 25 ml TES (Tris 50 mM, EDTA 1 mM,saccharose 7%). After adding 150 μg of lysostaphin, the mixture wasincubated 30 min at 37° C. Then, 2 ml of SDS 20% and 6 ml of EDTA 0.25 Mwere added and the suspension was incubated 15 min at 37° C. 8 ml ofNaCl 5M were added and the mixture was kept 90 min at +4° C. After 30min centrifugation at 8000 rpm, the supernatent was incubated 15 min at37° C. with 5 μg of Rnase (Boehringer). 10 μg of Proteinase K were addedand the suspension was incubated 15 min at 65° C. DNA was precipitatedusing isopropanol (0.6 V for 1 V of DNA solution). After 30 mincentrifugation at 8000 g, the pellet was washed with 10 ml ethanol 70%.The washed DNA was dried at 56° C., dissolved in 10 ml water andpurified by dye-buoyant density centrifugation (ethidium bromide-cesiumchloride). The extrachromosomal band was collected. After removingethidium bromide, the solution of plasmid DNA was dialyzed using TEbuffer (Tris, 10 mM, EDTA 1 mM, pH 7).

Example 7 Plasmid DNA Isolation from E. coli

Cf. QIAfilter plasmid maxi protocol for large-scale preparations andQIAprep Spin plasmid kit protocol for mini-preparations.

Quiagen GmbH and Quiagen Inc. (Hilden, Germany)

-   -   Plasmid maxi kit    -   Ref: 12262    -   Miniprep kit    -   Ref: 27104

Example 8 Transformation by Electroporation of the S. aureus RecipientStrain, RN4220

1—Preparation of Cells

200 ml of BHI was inoculated with 20 ml of an overnight culture ofRN4220 (Kreiswirth et al., Nature 1983, 306:709-712) and incubated at37° C. with shaking. When the OD reached 0.4 at 600 nm, the suspensionwas kept in ice. The pellet was washed three times with 20 ml of coldHepes buffer (saccharose 9.31%-Hepes 0.19%-pH. 7.4). The pellet wasresuspended in 2.5 ml of Hepes buffer containing 10% glycerol. Aliquotsof 100 μl cell suspension (3.10¹⁰/ml) were stored at −80° C.

2—Electroporation

After thawing at room temperature, the 100 μl aliquot of cells was keptin ice. After adding 10 μl of a solution containing 1 μg of plasmid DNA,the mixture was transferred to a cold 0.2 cm electroporation cuvette.The Gene Pulser (BioRad) was set at 25 uF and 2.5 KV and the PulseController to 100 Ω. This produced a pulse with a constant time of 2.3to 2.5 m sec. The cuvette was removed from the chamber and 1 ml of SOC(2% bactotryptone, 0.5% bactoyeast extract, 10 mM NaCl, 2.5 mMKCl, 10 mMMgCl₂, 10 mM MgSO₄, 20 mM glucose) was added. The cell suspension wastransferred in a propylene tube and incubated with shaking at 37° C. for1 hr. The suspension was then plated on selective medium, whichconsisted of BHIA containing 10 Mg/ml erythromycin or 10 μg/ml of PIIA.The plates were incubated 48 h at 37° C. and the transformants isolatedon selective medium. The further studies were carried out on a singleisolated colony.

Example 9 Polymerase Chain Reaction

DNA was amplified by PCR in a Crocodile 11 thermal cycler (Appligène)with approximately long of cellular DNA or 1 ng of plasmid DNA. Thereaction mixture contained 0.6 μM of each oligonucleotide serving asprimer, 200 μM of each deoxynucleotide triphosphate, 2.5 U of Taq DNAPolymerase (Amersham, Int.), and 1× buffer (Amersham, Int.). The finalreaction volume was adjusted to 100 μl with H₂O and the sample was thencovered by 50 μl of heavy white mineral oil (Sigma Chemical Co, St.Louis, Mo.).

PCR experiments were carried out at high or low stringency, depending onthe primers used. At high stringency, the PCR was performed with aprecycle of 3 min at 95° C. and 2 min at 60° C., 30 cycles of 20 sec at72° C., 20 sec at 95° C., 20 sec at 60° C. followed by a cycle of 1 minat 72° C. At low stringency, the PCR was performed with a precycle of 5min at 95° C., 35 cycles of 2 min at 40° C., 1 min 30 sec at 72° C., 30sec at 95° C. followed by a cycle of 4 min at 40° C. and 12 min at 72°C. The oligonucleotides used at high stringency are indicated in theTable below. PRIMER vgaB Oligo I 5′-AAGTCGACTGACAATATGAGTGGTGG-3′                  SalI Oligo II 5′-CTGCAGATGCCTCAACAGCATCGATATCC-3′              PstI vatC Oligo III 5′-ATGAATTCGCAAATCAGCAAGG-3′     EcoRIOligo IV 5′-TCGTCTCGAGCTCTAGGTCC-3′ SacI vgbB Oligo V5′-CAGCAGTCTAGATCAGAGTGG-3′     XbaI Oligo VI 5′-CATACGGATCCACCTTTTCC-3′ BamHl

Example 10 Labelling of DNA Probes

Plasmid DNA was labelled with [α-³²P]dCTP (110 Tbq mmol⁻¹) by the randomprinting technique using the Megaprime DNA labelling system (Amersham).

Example 11 Blotting and Hybridization

Hybond-N+ membranes (Amersham) were used for blotting. DNA wastransferred from agarose gels to the membranes by the capillary blottingmethod of Southern Blotting. DNA was denatured and fixed to themembranes according to the protocol described in the handbook user ofHybond-N+ membranes.

Prehybridization and hybridization were done at 68° C. in a mixturecontaining 5× SSPE (1× SSPE is 0.3 M NaCl, 30 m tri-sodium citrate), 5×Denhardt's solution, 0.5% (w/v) SDS, and 100 μg ml⁻¹ salmon sperm DNA.The membranes containing DNA transferred from agarose gels were treatedwith 10 ng ml⁻¹ radiolabeled DNA probe. Washing was started with twosuccessive immersions in 2× SSPE, 0.1% SDS, at room temperature for 10min, followed by one immersion in 1× SSPE, 0.1% SDS, at 68° C. for 15min, and finally by one immersion in 0.1× SSPE, 0.1% SDS, at 68° C. for15 min. The washed blots treated with the radiolabeled probe wereexposed to Fuji RX film at −70° C.

Example 12 Nucleotides Sequence Determination

For vatC and vgbB, the sequencing reaction was performed by PCRamplification in a final volume of 20 μl using 500 ng of plasmid DNA,5-10 pmoles of primer and 9.5 μl of DyeTerminators premix according toApplied Biosystems protocol. After heating to 94° C. for 2 min, thereaction was cycled as the following: 25 cycles of 30 s at 94° C., 30 sat 55° C., and 4 min at 60° C. (9600 thermal cycler Perkin Elmer).Removal of excess of DyeTerminators were performed using Quick Spincolumns (Boehringer Mannheim). The samples were dried in a vacuumcentrifuge and dissolved with 411 of deionized formamide EDTA pH 8.0(5/1). The samples were loaded onto an Applied Biosystems 373A sequencerand run for 12 h on a 4.5% denaturing acrylamide gel.

Primers used for sequencing the following genes: vatC5′-GAAATGGTTGGGAGAAGCATACC-3′ 5′-CAGCAATCGCGCCCGTTTG-3′5′-AATCGGCAGAATTACAAACG-3′ 5′-CGTTCCCAATTTCCGTGTTACC-3′ vgbB5′-GTTTCTATGCTGATCTGAATC-3′ 5′-GTCGTTTGTAATTCTGCCGATT-3′5′-GGTCTAAATGGCGATATATGG-3′ 5′-TTCGAATTCTTTTATCCTACC-3′

For vgaB, DNA was sequenced according to the instructions provided bythe T7Sequencing™ kit from Pharmacia Biotech (Uppsala, Sweden),procedures C and D.

Primers used for sequencing the following genes: vgaB5′-GCTTGGCAAAAGCAACC-3′ 5′-TGAATATAGGATAG-3′ 5′-TTGGATCAGGGCC-3′5′-CAATTAGAAGAACCAC-3′ 5′-CAATTGTTCAGCTAGG-3′ 5′-GAATTCATTCTATGG-3′5′-TACACCATTGTTACC-3′ 5′-CAAGGAATGATTAAGCC-3′ 5′-GATTCAGATGTTCCC-3′5′-TCATGGTCGCAATG-3′ 5′-GTTGCTTTCGTAGAAGC-3′ 5′-GTTATGTCATCCTC-3′5′-GGTTCATCTACGAGC-3′ 5′-GGATATCGATGCTG-3′ 5′-GCCAACTCCATTC-3′5′-CCTAGCTGAACAATTG-3′ 5′-GAAGGTGCCTGATCC-3′ 5′-ATACTAGAAATGC-3′

Example 13 DNA Cloning

A standard protocol was followed for cloning into the vector p0X7, alsonamed pOX300, the 2.4 kb HindIII-HaeIII fragment of pIP1633 carryingvgaB (FIG. 1) and the plasmid pIP1714 carrying vatC and vgbB (FIG. 4),linearized by cleavage with HindIII. The vector DNA (10-20 μg) and theplasmids used in cloning experiments were cleaved with the appropriaterestriction enzymes (30 Units) and purified by GeneClean Kit (Bio 101,La Jolla, Calif.). To avoid religation, the vector cleaved with a singleenzyme was dephosphorylated by 30 min incubation at 37° C. with 5 Unitsof alkaline phosphatase. Ligation was carried out in a total reactionvolume of 10 μl containing 0.1 μg of the vector, 0.1 μg of the plasmid,0.5 mM ATP, 1× T4 DNA ligase buffer and 0.1 Weiss Unit of T4 DNA ligase.After overnight incubation at 16° C., 1 to 2 μl of the ligation mixtureare used for transforming competent E. coli and the transformants wereselected on solid media containing 100 μg/ml of ampicillin.

Example 14 Susceptibility to Antimicrobial Agents

Susceptibility to antimicrobial agents was determined with a diskdiffusion assay and commercially available disks (Diagnostic Pasteur).Additional disks prepared in our laboratory contained streptogramin A(20 μg) or streptogramin B (40 μg)

-   -   NCCLS: Performance standards for antimicrobial disk        susceptibility test, 1984, Approved standard M2-A3, 4:369-402.    -   ECCLS: Standard for antimicrobial susceptibility testing by        diffusion methods, 1985, ECCLS Document, 5:4-14.

Minimal inhibitory concentrations (MICs) of antibiotics were determinedby serial twofold dilutions of antibiotics in MHA (Ericson H. M. and S.C. Sherris, ActaPathol. Microbiol. Scand., 1971, Suppl. 217: Section B).

Despite the relatively low frequency of detection of SgA^(R)staphylococci (1-10%) (Loncle et al., 1993; Allignet et al., 1996), fourgenes encoding resistance to streptogramin A have been detected andother resistance gene(s) are suspected to be carried by staphylococci.Surprisingly, the present and previous studies (Allignet et al., 1996)indicate that staphylococcal plasmids carrying two genes encodingstreptogramin A resistance by two distinct mechanisms (inactivation byacetyltransferases and increased efflux) are widespread amongstaphylococci (32 of the 48 plasmids investigated).

REFERENCES

The following publications have been cited herein. The entire disclosureof each publication is relied upon and incorporated by reference herein.

-   Allignet, J., Loncle, V., Mazodier, P. and El Solh, N. (1988)    Nucleotide sequence of a staphylococcal plasmid gene, vgb, encoding    a hydrolase inactivating the B components of virginiamycin-like    antibiotics. Plasmid 20,271-275.-   Allignet, J., Loncle, V. and El Solh, N. (1992) Sequence of a    staphylococcal plasmid gene, vga, encoding a putative ATP-binding    protein involved in resistance to virginiamycin A-like antibiotics.    Gene 117, 45-51.-   Allignet, J., Loncle, V., Simenel, C., Delepierre, M. and El    Solh, N. (1993) Sequence of a staphylococcal gene, vat, encoding an    acetyltransferase inactivating the A-type compounds of    virginiamycin-like antibiotics. Gene 130, 91-98.-   Allignet, J. and El Solh, N. (1995) Diversity among the    Gram-positive acetyltransferases inactivating streptogramin A and    structurally related compounds, and characterization of a new    staphylococcal determinant, vatB. Antimicrob. Agents Chemother. 39,    2027-2036.-   Allignet, J., Aubert, S., Morvan, A. and El Solh, N. (1996)    Distribution of the genes encoding resistance to streptogramin A and    related compounds among the staphylococci resistant to these    antibiotics. Antimicrob. Agents Chemother. 40, 2523-2528.-   Allignet, J. and El Solh, N. (1996) Sequence of a staphylococcal    plasmid gene vga B, encoding a putative ATP-binding protein related    to vga involved ii resistance to streptogramin A, 8th International    Symposium on Staphylococci and Staphylococcal Infections, 23-26    Jun., 1996, p. 202, 239.-   Aumercier, M., Bouhallab, S., Capmau, M. L. and Le Goffic, F. (1992)    RP59500: a proposed mechanism for its bactericida. activity. J.    Antimicrob. Chemother. 30, 9-14.-   Barrasa, M. i., Tercero, J. A., Lacalle, R. A. and    Jimenez, A. (1995) The ardl gene from Streptomyces capreolus encodes    a polypeptide of the ABC-transporters superfamily which confers    resistance to the amino-nucleotide antibiotic A201A. Eur. J.    Biochem. 228, 562-569.-   Blanc, V., Salah-Bey, K., Folcher, M. and Thompson, C. J. (1995)    Molecular characterization and transcriptional analysis of a    multidrug resistance gene cloned from the pristinamycin-producing    organism, Streptomyces pristinaespiralis. Mol. Microbiol. 17,    989-999.-   Cocito, C. (1979) Antibiotics of the virginiamycin family,    inhibitors which contain synergistic components. Microbiol. Rev. 43,    145-198.-   Di Giambattista, M., ChinaIi, G. and Cocito, C. (1989) The molecular    basis of the inhibitory activities of type A and type B    synergimycins and related antibiotics on ribosomes. J. Antimicrob.    Chemother. 24, 485-507.-   Dyke, K. G. H. and Curnock, S. P. (1989) The nucleotide sequence of    a small crypticplasmid found in Staphylococcus aureus and its    relationship to other plasmids. FEMS Microbiol. Lett. 58, 209-216.-   El Solh, N., Fouace, J. M., Shalita, Z., Bouanchaud, D. H.,    Novick, R. P. and Chabbert, Y. A. (1980) Epidemiological and    structural studies of Staphylococcus aureus R plasmids mediating    resistance to tobramycin and streptogramin. Plasmid 4, 117-120.-   Entenza, J. M., Drugeon, H., Glauser, M. P. and Moreillon, P. (1995)    Treatment of experimental endocarditis due to    erythromycin-susceptible or—resistant methicillin—resistant    Staphylococcus aureus with RP59500. Antimicrob. Agents Chemother.    39, 1419-1424.-   Fantin, B., Leclercq, R., Merl, Y., Saint-Julien, L., Veyrat, C.,    Duval, J. and Carbon, C. (1995) Critical influence of resistance to    streptogramin B-type antibiotics on activity of RP59500    (quinupristin-dalfopristin) in experimental endocarditis due to    hylococcus aureus. Antimicrob. Agents Chemother. 39, 400-405.-   Fierro, J. F., Vilches, C., Hardisson, C. and Salas, J. A. (1989)    Streptogramins—inactivating activity in three producer    streptomycetes. FEMS Microbiol. Lett. 58, 243-246.-   Geistlich, M., Losick, R., Turner, J. R. and Rao, R. N. (1992)    Characterization of a novel regulatory gene governing the expression    of a polyketide synthase gene in Streptomyces ambofaciens. Mol.    Microbiol. 6, 2019-2029.-   Griswold, M. W., Lomaestro, B. M. and Briceland, L. L. (1996)    Quinupristin-dalfopristin (RP59500)—an injectable streptogramin    combination. Amer. J. Health-Syst. Pharm. 53, 2045-2053.-   Hyde, S. C., Emsley, P., Hartshorn, M. J., Mimmack, M. M., Gileadi,    U., Pearce, S. R., Gallagher, M. P., Gill, D. R., Hubbard, R. E. and    Higgins, C. F. (1990) Structural model of ATP-binding proteins    associated with cystic fibrosis, multidrug resistance and bacterial    transport. Nature 346, 362-365.-   Kim, C. H., Otake, N. and Yonehara, H. (1974) Studies on mikamycin B    lactonase. I. Degradation of mikamycin B by Streptomyces    mitakaensis. J. Antibiot. 27, 903-908.-   Kloos, W. E. and Schleifer, K. H. (1986). Genus IV. Staphylococcus    Rosenbach 1884. 18AL, (Nom. Cons. ( )pin. 17 Jud. Comm. 1958, 153).    In: Sneath, P. H. A., Mair, N. S., Sharpe, M. E. and Holt, J. G.    (Eds.), Bergey's manual of systematic bacteriology. Williams &    Wilkins, Baltimore, Vol. 2, pp. 1013-103.-   Kreiswirth, B. N., Lofdahl, S., Bethey, M. J., O'Reilly, M.,    Shlievert, P. M., Bergdoll, M. S. and Novick, R. P. (1983) The toxic    shock exotoxin structural gene is not detectably transmitted by a    prophage. Nature 306, 709-712.-   Kyte, J. and Doolittle, R. F. (1982) A simple method for displaying    the hydropathic character of a protein. J. Mol. Biol. 157, 105-132.-   Liassine, N., Allignet, J. Morvan, A., Aubert, S. and El    Solh, N. (1997) Multiplicity of the genes and plasmids conferring    resistance to pristinamycin in Staphylococci selected in an Algerian    hospital, Zbl. Bakt. 1212.-   Loncle, V., Casetta, A., Buu-Ho•, A. and El Solh, N. (1993) Analysis    of pristinamycin-resistant Staphylococcus epidermidis isolates    responsible for an outbreak in a parisian hospital. Antimicrob.    Agents Chemother. 37, 2159-2165.-   MacLaughlin, J. R., Murray, C. L. and Rabinowitz, C. (1981) Unique    features in the ribosome binding site sequence of the Gram-positive    Staphylococcus aureus §-lactamase gene J. Biol. Chem. 256,    11283-11291.-   Meyer, C., Bierbaum, G., Heidrich, C., Reis, M., SŸling, J.,    Iglesias-Wind, M., Kempter, C., Molitor, E. and Sahl, H.-G. (1995)    Nucleotide sequence of the antibiotic Pep5 biosynthetic gene cluster    and functional analysis of PepP and PepC: Evidence for a role of    PepC in thioether formation. Eur. J. Biochem. 232, 478-489.-   Moran, C. P., Jr., Lang, N., LeGrice, S. F. J., Lee, G., Stephens,    M., Sonenshein, A. L., Pero, J. and Losick, R. (1982) Nucleotide    sequences that signal the initiation of transcription and    translation in Bacillus subtilis. Mol. Gen. Genet. 186, 339-346.-   Olano, C., Rodriguez, A. M., Mndez, C. and Salas, J. A. (1995) A    second ABC transporter is involved in oleandomycin resistance and    its secretion by Streptomyces antibioticus. Mol. Microbiol. 16,    333-343.-   Peschke, U., Schmidt, H., Zhang, H.-Z. and Piepersberg, W. (1995)    Molecular characterization of the lincomycin-production gene cluster    of Streptomyces lincolnensis 78-11. Mol. Microbiol. 16, 1137-1156.-   Rende-Fournier, R., Leclercq, R., Galimand, M., Duval, J. and    Courvalin, P. (1993) identification of the satA gene encoding a    streptogramin A acetyltransferase in Enterococcus faecium BM4145.    Antimicrob. Agents Chemother. 37, 2119-2125.-   Ross, J. I., Eady, E. A., Cove, J. H., Cunliffe, W. J., Baumberg, S.    and Wootton, J. C. (1990) Inducible erythromycin resistance in    staphylococci is encoded by a member of the ATP-binding transport    super-gene family. Mol. Microbiol. 4(7), 1207-1214.-   Ross, J. I., Eady, E. A., Cove, J. H. and Baumberg, S. (1995)    Identification of a chromosomally encoded ABC-transport system with    which the staphylococcal erythromycin exporter MsrA may interact.    Gene 153, 93-98.-   Ross, J. I., Eady, E. A., Cove, H. H. and Baumberg, S. (1996)    Minimal functional system required for expression of erythromycin    resistance by MSRA in Staphyloocccus aureus RN4220. Gene 183,    143-148.-   Rosteck, P. R. J., Reynolds, P. A. and Hershberger, C. L. (1991)    Homology between proteins controlling Streptomyces fradiae tylosin    resistance and ATP-binding transport.

Gene 102, 27-32.

-   Sanger, F., Nicklen, S. and Coulson, A. R. (1977) DNA sequencing    with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,    5463-5467.-   Schoner, B., Geistlich, M., Rosteck, P. R., Jr., Rao, R. N., Seno,    E., Reynolds, P., Cox, K., Burgett, S. and Hershberger, C. (1992)    Sequence similarity between macrolide-resistance determinants and    ATP-binding transport proteins. Gene 115, 93-96.-   Southern, E. M. (1975) Detection of specific sequences among DNA    fragments separated by gel electrophoresis. J. Mol. Biol. 98,    503-517.-   Tinoco, I., Jr., Borer, P. N., Dengler, B., Levine, M. D.,    Uhlenbeck, O. C., Crothers, D. M. and Gralla, J. (1973) Improved    estimation of secondary structure in ribonucleic acids. Nature New    Biol. 246, 40-41.-   Torralba, M. D., Frey, S. E. and Lagging, L. M. (1995) Treatment of    methicillin-resistant Staphylococcus aureus infection with    quinupristin dalfopristin. Clin. Infect. Dis. 21, 460-461.-   von Heijne, G. (1986) A new method for predicting signal sequence    cleavage sites. Nucl. Acids Res. 14, 4683-4690.-   Walker, J. E., Saraste, M., Runswick, M. J. and Gay, N. J. (1982′    Distantly related sequences in the a- and §-subunits of ATP    synthase, myosin, kinases and other ATP-requiring enzymes and a    common nucleotide binding fold. EMBO J. 1 945-951.-   Watson, M. E. E. (1984) Compilation of published signal sequences.    Nucl. Acids Res. 12, 5145-5148.

1-36. (canceled)
 37. A purified peptide encoded by a polynucleotideselected from: a) SEQ ID NO:1 or a fragment of SEQ ID NO:1 containing 15to 40 nucleotides; b) SEQ ID NO:2 or a fragment of SEQ ID NO:2containing 15 to 40 nucleotides; c) SEQ ID NO:3 or a fragment of SEQ IDNO:3 containing 15 to 40 nucleotides; d) SEQ ID NO:11; e) SEQ ID NO:12;f) SEQ ID NO:13; and g) SEQ ID NO:14.
 38. The purified peptide accordingto claim 37, wherein the purified peptide is encoded by SEQ ID NO:1. 39.The purified peptide according to claim 37, wherein the purified peptideis encoded by a fragment of SEQ ID NO:1 containing 15 to 40 nucleotides.40. The purified peptide according to claim 37, wherein the purifiedpeptide is encoded by SEQ ID NO:2.
 41. The purified peptide according toclaim 37, wherein the purified peptide is encoded by a fragment of SEQID NO:2 containing 15 to 40 nucleotides.
 42. The purified peptideaccording to claim 37, wherein the purified peptide is encoded by SEQ IDNO:3.
 43. The purified peptide according to claim 37, wherein thepurified peptide is encoded by a fragment of SEQ ID NO:3 containing 15to 40 nucleotides.
 44. The purified peptide according to claim 37,wherein the purified peptide is encoded by SEQ ID NO:11.
 45. Thepurified peptide according to claim 37, wherein the purified peptide isencoded by SEQ ID NO:12.
 46. The purified peptide according to claim 37,wherein the purified peptide is encoded by SEQ ID NO:13.
 47. Thepurified peptide according to claim 37, wherein the purified peptide isencoded by SEQ ID NO:14.
 48. A purified peptide selected from: a) SEQ IDNO:4 or a fragment of SEQ ID NO:4 containing at least 10 amino acids; b)SEQ ID NO:5 or a fragment of SEQ ID NO:5 containing at least 10 aminoacids; c) SEQ ID NO:6 or a fragment of SEQ ID NO:6 containing at least10 amino acids; d) SEQ ID NO:7; e) SEQ ID NO:8; f) SEQ ID NO:9; and g)SEQ ID NO:10.
 49. The purified peptide according to claim 48, whereinthe purified peptide is SEQ ID NO:4.
 50. The purified peptide accordingto claim 48, wherein the purified peptide is a fragment of SEQ ID NO:4containing at least 10 amino acids.
 51. The purified peptide accordingto claim 48, wherein the purified peptide is SEQ ID NO:5.
 52. Thepurified peptide according to claim 48, wherein the purified peptide isa fragment of SEQ ID NO:5 containing at least 10 amino acids.
 53. Thepurified peptide according to claim 48, wherein the purified peptide isSEQ ID NO:6.
 54. The purified peptide according to claim 48, wherein thepurified peptide is a fragment of SEQ ID NO:6 containing at least 10amino acids.
 55. The purified peptide according to claim 48, wherein thepurified peptide is SEQ ID NO:7.
 56. The purified peptide according toclaim 48, wherein the purified peptide is SEQ ID NO:8.
 57. The purifiedpeptide according to claim 48, wherein the purified peptide is SEQ IDNO:9.
 58. The purified peptide according to claim 48, wherein thepurified peptide is SEQ ID NO:10.
 59. A purified polypeptide encoded bya nucleotide sequence selected from vgaB, vgbB, and vatC.
 60. A purifiedpolypeptide or a peptide fragment having at least 10 amino acids, whichis recognized by antibodies that specifically bind to a purified peptideaccording to claim
 37. 61. A purified polypeptide or a peptide fragmenthaving at least 10 amino acids, which is recognized by antibodies thatspecifically bind to a purified peptide according to claim 48.